The present invention relates to a composition comprising at least one photosensitizer and a solid carrier, wherein the photosensitizer is chemically bonded to said carrier and wherein said carrier is swellable in water, and to a process for combating bacterial germs, algae, yeast and fungi in water.
Over the years, water purity and its influence on the products in contact with the water has gained increasing importance. One of the greatest problems in providing clean water is bacterial contamination and contamination due to algae, yeast and fungi. There are numerous processes for preventing or removing such contamination from water, but all have certain disadvantages. Thus, for example the prior art includes processes in which chemicals, steam, UV light, sterile filtration or ozone are employed for disinfection. All these processes have disadvantages, however, which in some cases makes use on a large scale seem not very appropriate economically. Thus, for example, treatment with chemicals can adversely influence the quality of the water due to the chemicals introduced. Ultraviolet treatment involves the risk that disinfection is only partially carried out, especially in the case of relatively large amounts of water to be treated. Although sterile filtration is capable of eliminating bacterial germs to a certain degree, it is unsuitable for preventing renewed bacterial growth.
One field of use which necessitates sterilization of large quantities of water is fish farming, which in particular should also satisfy the increasing foodstuff requirements. The productivity of fish farms depends, inter alia, on the extent to which transmission of diseases to the fish being bred can be limited or prevented. Contamination of the water with germs, algae, yeast and fungi is particularly severe during seasons when the temperature is increased and bacterial growth and growth of algae, yeast and fungi thus rises. The stress conditions for the fish also increase because of reduced concentrations of dissolved oxygen, so that their susceptibility to disease increases.
In monocultures, such as fish farms, the large number of animals per unit volume of water increases the multiplication and spread of potentially pathogenic microorganisms which can infect the fish. The germ content of the water can thus adversely influence the productivity of the unit.
The terms xe2x80x9cgerms and/or microorganismsxe2x80x9d relate to microbes especially microbes which can be pathogeous, as e.g. gram-positive, gram-negative bacteriae, algae, yeast and fungi, wherein said germs can be present alone or in combination with other microorganisms.
Numerous bacterial pathogens have been identified in water, including Gram-positive rod-shaped and coccal bacteria, aeromonads, myxobacteria, Gram-negative rod-shaped bacteria, vibrios and pseudomonads. In addition to the above-mentioned processes for sterilizing water, vaccines or the introduction of antimicrobial compounds have also been resorted to in particular as measures to suppress the spread of diseases. However, these measures are of limited efficacy and result in enormous costs, and furthermore represent a risk to the environment.
Ozone treatment of water is currently the most important process for sterilizing water. However, this process requires removal of the ozone from the treated water after conclusion of the ozone treatment, the removal of the ozone necessitating a high expenditure on apparatus.
Thus, for example, frequently used vaccines, especially inactivated bacterial cells or purified subcellular organelles, are often harmful to numerous constituents of sea water or fresh water systems, with the result that the ecological equilibrium system is disturbed. Similarly, antimicrobial substances, from antibiotics to various other chemicals, also have several disadvantages, such as, for example, the development of a resistant microflora and the transmission of resistance to other pathogens by means of plasmid transfer; release of active compounds into the water of the environment with possibly adverse effects on humans; deposition of degradation products of the chemicals introduced in the environment with possibly accumulating toxic effects on the organisms in the vicinity of the unit. Typical example of environmentally harmful chemicals which are used in the fish industry are chloramphenicol, chloramine B and T and tetracyclines.
U.S. Pat. No. 4,530,924 discloses a process for combating micro-organisms in or on organic or inorganic substrates. EP-A-0 054 992 describes bleach compositions containing a photo-activator. WO93/00005 refers to a method for inactivating pathogens in a body fluid. U.S. Pat. No. 4,648,992 and U.S. Pat. No. 4,465,452 describe water-soluble phthalocyanine compounds for bleaching textiles. WO93/00815 mentions compositions of a polymer and a photosensitizer having autosterile character on exposure to visible light.
The present invention is accordingly based on the technical problem of providing a composition for combating bacterial germs, algae, yeast and fungi in water which has a high efficiency, preserves the environment and also can be carried out inexpensively on a large industrial scale.
This problem is solved according to the invention by a composition comprising at least one photosensitizer of the tetrapyrrole and/or tetraazapyrrole series and a solid carrier, characterized in that the photosensitizer is chemically bonded to said carrier and said carrier is swellable in water.
The present invention also relates to a process for combating bacterial germs, algae, yeast and fungi in water, wherein the germ-containing water is brought into contact with the composition according to the invention and is subjected to electromagnetic radiation.
Moreover, the present invention relates to the use of the above-mentioned composition for the treatment of water containing bacterial germs, algae, yeast and/or fungi.
FIGS. 1-13 show the action of photosensitizers on various microorganisms as explained below.
FIG. 14 shows a unit for treating water of a fish farm.
FIG. 1 shows the effect of irradiation with visible light on the survival of V. anguillarum cells after incubation for 5 minutes with 10 xcexcg/ml of tetra(4-N-methyl-pyridyl)porphine T4MPyP and various washing operations; ▪0 washing operation, xe2x97xaf1 washing operation, ▾3 washing operations.
FIG. 2 shows the effect of irradiation with visible light on the survival of E. coli cells after incubation for 5 minutes with 10 xcexcg/ml of T4MPyP and various washing operations; ▪0 washing operation, xe2x97xaf1 washing operation, ▾3 washing operations.
FIG. 3 shows the effect of irradiation with visible light on the survival of V. anguillarum cells after incubation for 5 minutes with di(4NMPy)Ph2P (8.4 xcexcM) in aqueous suspension, xe2x96xa10 washing operation, ∘1 washing operation, xcex943 washing operations.
FIG. 4 shows the effect of irradiation with visible light on the survival of E. coli cells after incubation for 5 minutes with di(4NMPy)Ph2P (8.4 xcexcM) in aqueous suspension, xe2x96xa10 washing operation, ∘1 washing operation, xcex943 washing operations.
FIG. 5 shows the effect of HP concentration in the incubation medium on the amount of porphyrin bound by cells of M. hominis (xe2x97xaf,∘), A. laidlawii+S(▪,∘) and A. laidlawiixe2x88x92S (▾,xcex94) in the exponential (filled symbols) and stationary (open symbols) phases of growth. The incubation was performed at room temperature for 30 min and the cell-bound HP was estimated by a spectrophotofluorimetric procedure.
FIGS. 6 and 7 show the survival curves of C. albicans cells, obtained from brain heart broth, irradiated at different temperatures in the presence of 1 xcexcg HP mlxe2x88x921 and plated on Sabouraud agar (FIG. 6) and brain heart agar (FIG. 7). Vertical bars indicate standard error.
FIG. 8 shows the survival of mycoplasma cells in the exponential (filled symbols) and stationary (open symbols) phases of growth after incubation with HP for 90 min in the dark. The initial cell concentrations [log(c.f.u.mlxe2x88x921] were 7 for M. hominis and 6 for A. laidlawii (+S and xe2x88x92S). Each point represents the mean of three independent experiments performed in duplicate. The largest standard error obtained [log(c.f.u.mlxe2x88x921] was 0.18; xe2x97xaf, ∘, M. hominis; ▪, xe2x96xa1, A. laidlawii+S, ▾, xcex94 A. laidlawiixe2x88x92S.
FIG. 9 shows the time-dependence of cell survival upon visible light irradiation at 37xc2x0 C. of M. hominis (xe2x97xaf), A. laidlawii+S (▪), A. laidlawiixe2x88x92S (▾) in the exponential phase of growth, in the presence of 0.1 xcexcg HP mlxe2x88x921 the cells (initial concentrations as for FIG. 8) had been previously incubated with HP for 60 min in the dark at room temperature. Each point represents the mean of four independent experiments performed in duplicate. The largest standard error obtained [log(c.f.u.mlxe2x88x921] was 0.2.
FIG. 10 shows the survival curves of C. albicans cells, obtained from brain heart broth, exposed to visible light at 37xc2x0 C. in the presence of 0.1 (∘), 1 (xe2x96xa1) and 10 (xcex94) xcexcg HP mlxe2x88x921 and plated on brain heart agar. Vertical bars indicate standard error.
FIG. 11 shows photoinhibition of the marine alga Phaeodactylum tricomutum (diatomea) using as photosensitizer the dicationic porphyrin, meso-diphenyl-di(N-methyl-pyridyl)porphine (abbreviated as Di(4NMPy)Ph2P) with a porphyrin concentration of 8.4 xcexcM; medium: aqueous solution at pH 7.4 containing 2.4xc3x97105 algae/ml; temperature: 16-18xc2x0 C. (before and during irradiation); light source: full spectrum visible light (filament lamps), 10 mW/cm2; protocol: after irradiation, the system was kept in the dark and incubated at 18xc2x0 C.; viability counts were carried out at selected post-irradiation times; control: algae kept in the dark at 18xc2x0 C.; light only: algae specimens irradiated in the absence of photosensitizer for 30 min.
The meanings of the symbols are:
OD650: optical density at 650 nm (absorption)
xe2x80x94xe2x96xa1xe2x80x94control
xe2x80x94∘xe2x80x94+30 min light
xe2x80x94xcex94xe2x80x94+porphyrin-light
xe2x80x94▴xe2x80x94+porphyrin+min light
xe2x80x94⋄xe2x80x94+porphyrin+15 min light
xe2x80x94+xe2x80x94+porphyrin+30min light
FIG. 12 shows an experiment which was carried out with a freshwater alga, namely a Clamydomonas sp., in the same manner used for the algae from a marine water as described above.
The evaluation of the legal growth at about 430 h after the end of the irradiation studies (post irradiation incubation) is shown:
xe2x80x94xe2x96xa1xe2x80x94control (untreated algae):
xe2x80x94∘xe2x80x94+Irradiated 30 min. in the absence of porphyrin:
xe2x80x94xcex94xe2x80x94+Incubated with porphyrin in the dark:
xe2x80x94▴xe2x80x94+Irradiated with porphyrin for 1 min.:
xe2x80x94⋄xe2x80x94Irradiated with porphyrin for 15 min.:
xe2x80x94+xe2x80x94+Irradiated with porphyrin for 30 min.:
The numbers are proportional to the number of algae per volume unit (ml).
FIG. 13 shows the effect of visible light irradiation on cell survival of E. coli after 5 min incubation with 10 xcexcg/ml of different porphyrins and without washing; ▪ tetraphenylporphine sulfonate (TPPS4), xe2x97xaf tetra(4-N-methyl-pyridyl)porphine (T4MPyP), ▾ tetra(N,N,N-trimethyl-anilinium)porphine (T4MAP).
FIG. 14 shows a construction, given by way of example, of a unit for treatment of water using the process according to the invention, which unit can be modified in any desired manner by the different use of technical components according to the prior art available and the aim of its use.